Herpes simplex viruses 1 and 2 (HSV-1, HSV-2) cause a wide range of significant diseases, including oral/genital lesions, corneal blindness and encephalitis. HSV-1 and HSV-2 are both transmitted sexually and can infect babies during birth. Therapy for HSV relies primarily on nucleoside analogs such as acyclovir (ACV) which are insufficiently effective, and resistant viruses are becoming common among immunosuppressed persons. Therefore, there is an urgent need for better anti-herpesvirus drugs. The nucleotidyl transferase superfamily (NTS) of enzymes comprises a range of nucleases and recombinases that are essential for DNA replication, recombination, and nucleic acid turnover. Our screening program to identify inhibitors of HSV replication demonstrated that a set of NTS enzyme inhibitors have profound anti-HSV activity in vitro without significant cytotoxicity. They also suppress replication of ACV-resistant mutant strains. In this STTR project, we will execute a Hit-to-Lead program based on an initial set of highly active, small- molecule confirmed hits against HSV. An established preliminary SAR will be the starting platform from which the project will begin the process of optimizing already potent inhibitors through in vitro evaluation and prioritization, and ultimately generate a lead series for Lead Optimization (LO). Aim 1. Iteratively produce enhanced Hit-To-Lead ?HT inhibitors of HSV replication. Compounds sets will be iteratively synthesized and tested for HSV replication inhibition, specificity (human RNaseH1 counter- screen), and cytotoxicity. Chemical design for LO to increase drug-like characteristics will be performed. Top hits will be tested for synergy with ACV. Aim 2: Profile in vitro ADME and toxicity parameters. ADME will be evaluated in mouse and human liver microsomes, and expanded toxicity assessments will be conducted in mouse and human cell types. Aim 3. Identify select Lead Compounds through further pre-clinical profiling and Lead Optimization. Biological profiling (Aims 1 and 2) in conjunction with evaluation of physico-chemical properties of promising derivatives will guide selection of Lead Compound(s) with high activity and low toxicity profiles. These will be advanced to further in vitro profiling in protein binding and Caco-2 permeability assays. Significant Impact: New therapies are urgently needed for patients who suffer from recurrent outbreaks of HSV infections, and especially immunocompromised individuals infected with nucleoside analog-resistant strains. This project will result in the generation of a lead series of highly active compounds suitable for LO efforts. The ultimate goal is directed at generating new therapeutic drug candidate(s) for the treatment of herpesvirus infections, including HSV-2, as monotherapies or combination therapies with existing drugs.